Attention recently has focused on the relationship between endothelial dysfunction and coronary artery disease (Widlansky, et al., J. Am. Coll. Cardiol., 42: 1149–60 (2003) and Okumura, et al., J. Am. Coll. Cardiol., 19: 752–8 (1992)). The term “endothelial dysfunction” refers to a broad class of alterations in endothelial phenotype that may lead to the development of atherosclerosis and other vascular abnormalities (Levine, et al., New Engl. J. Med., 332: 512–21 (1995)), loss of endothelium-dependent vasodilation, and increased expression of leukocyte chemotactic factors, adhesion molecules and inflammatory cytokines (Ruiz-Ortega, et al. Hypertension, 38: 1382–7 (2001)).
The methods available currently for studying endothelial function are based on indirect evaluation of vasoreactivity to different stimuli. Stimuli that increase the production of endothelium-derived nitric oxide have been proven useful in assessing endothelium-dependent vasodilation in humans, as has the measurement of vascular response to receptor-dependent agonists such as acetylcholine, bradykinin or substance P. However, important limitations associated with the detection of these vascular responses have to be considered, including the requirement for a local delivery of the agonist via an intra-arterial infusion. Such limitations prevent widespread use of such techniques and carry significant risk of complication. Other techniques in use currently for the assessment of endothelial function provide indirect measurement of the vasoreactivity of brachial arteries using plethysmography or vascular ultrasound. Although studies suggest that endothelium-dependent responses detected in the brachial artery correlate with coronary artery function, these techniques do not allow for direct evaluation of a specific vascular region and do not correlate with microvascular function (Eskurza, et al. Am. J. Cardiol., 88: 1067–9 (2001)).
Recent studies support the hypothesis that the interaction between the endothelium and microbubbles used for myocardial contrast echocardiography could form a basis for studying endothelial dysfunction. Normally microbubbles have kinetics similar to erythrocytes and pass unimpeded through the large vessels and coronary microcirculation (Jayaweera, et al. Circ. Res., 74: 1157–65 (1994)). However, in the presence of dysfunctional endothelium, transit of microbubbles is delayed despite normal blood flow (Villanueva, et al. J. Am. Coll. Cardiol., 30: 689–93 (1997)). Although previous studies have demonstrated that endothelial dysfunction results in retention of microbubbles in the microcirculation (Villanueva, et al. J. Am. Coll. Cardiol., 30: 689–93 (1997) and Christiansen, et al. Circulation, 105: 1764–7 (2002)), the value of using microbubbles to detect endothelial dysfunction by direct imaging or imaging of large vessels has not been evaluated to date.
Villanueva et al., in J. Am. Coll. Cardiol., 30: 689–93 (1997), evaluated the influence of the characteristics of the endothelial surface on albumin microbubble transit using an in vitro perfusion model of cultured coronary endothelial cells. This in vitro system demonstrated that microbubbles do not adhere to normal cells, but, during inflammatory conditions, there is binding of microbubbles to exposed extracellular matrix. In the same in vitro model, Villanueva, et al. demonstrated imaging of vascular endothelium using ICAM-binding microbubbles (see WO/99/13918). However, a non-invasive direct method to detect endothelial dysfunction of a specific vessel of interest or in large vessels has not been developed.
There is a need in the art for detecting dysfunction of the endothelium at an early time point to allow early therapeutic intervention to prevent vascular disease before significant damage occurs. The present invention satisfies this need in the art.